Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

Gonadotropins and Sex Steroid Hormones in Captive-Reared Small Yellow Croaker (Larimichthys polyactis) and Their Role in Female Reproductive Dysfunction.

The seed production of small yellow croaker (SYC) is constrained by reproductive dysfunction in captive-reared females. Reproductive dysfunction is closely linked to endocrine reproductive mechanisms. To better understand the reproductive dysfunction in captive broodstock, functional characterization of gonadotropins (GtHs: follicle stimulating hormone ß subunit, fshß; luteinizing hormone ß subunit, lhß; and glycoprotein α subunit, gpα) and sex steroids (17ß-estradiol, E2; testosterone, T; progesterone; P) was performed using qRT-PCR, ELISA, in vivo, and in-vitro assay. The pituitary GtHs and gonadal steroids levels were significantly higher in ripen fish of both sexes. However, changes in lhß and E2 levels in females were not significant in the developing and ripen stages. Furthermore, GtHs and steroids levels were lower in females compared to males throughout the reproductive cycle. In vivo administration of gonadotropin releasing hormone analogue (GnRHa) significantly increased the expression of GtHs in both dose- and time-related manners. The lower and higher doses of GnRHa led to successful spawning in male and female SYC, respectively. Sex steroids in vitro significantly inhibited the expression of lhß in female SYC. Overall, GtHs were shown to play a vital role in final gonadal maturation, while steroids promoted negative feedback in the regulation of pituitary GtHs. Lower levels of GtHs and steroids might be key components in the reproductive dysfunction of captive-reared female SYC.

Regulation of steroid production and key genes in catfish Heteropneustes fossilis using recombinant gonadotropins.

The two gonadotropins, FSH and LH, stimulate growth and development of the gonads through gonadal biosynthesis of steroid hormones and growth factors. To date, cDNA sequences encoding gonadotropin subunits have been isolated and characterized from a large number of fish species. Recently, we successfully cloned and characterized gonadotropins (LHß, FSHß, and GPα) from the pituitary glands of the catfish, Heteropneustes fossilis. In the present study, we describe herein the production of recombinant stinging catfish, H. fossilis (hf) FSH (rhfFSH) and LH (rhfLH) using the methylotrophic yeast P. pastoris expression system. We further explored the hypothesis that the recombinant gonadotropins can modulate the hypothalamus-pituitary-ovarian (HPO) axis genes (avt, it, gnrh2, kiss2, and cyp19a1a) and regulate their transcriptional profile and steroid levels in relation to their annual developmental stage during preparatory and pre-spawning phases under in-vitro conditions. We found that the different concentrations of recombinant rhfFSH and rhfLH significantly stimulated E2 levels in the preparatory and prespawning season, and also upregulated gonadal aromatase gene expression in a dose dependent manner. Our results demonstrate that the yeast expression system produced biologically active recombinant catfish gonadotropins, enabling the study of their function in the catfish.

Cloning of gonadotropin Gph-alpha, FSH-beta and LH-beta subunits and seasonal profiles of steroid hormones in wild-caught Nile perch, Lates niloticus.

The Nile perch (np; Lates niloticus) is a freshwater teleost species with a potential for aquaculture in freshwater surroundings. However, wild-caught breeders have persistently failed to spawn spontaneously in captivity. Cloning of the gonadotropin subunits and analysing seasonal variation in reproductive hormone levels for a 1-year period were done to gain knowledge on the physiological basis underlying the reproductive biology of np. The ß-follicle-stimulating hormone (FSH-ß) and ß-luteinizing hormone (LH-ß) subunits and their common α-glycoprotein (Gph-α) subunit were cloned using 3' and 5' RACE-PCR. The nucleotide sequences of the npgph-α, npfsh-ß, and nplh-ß subunits were 664, 580 and 675 nucleotides in length, encoding peptides of 124, 120 and 148 amino acids, respectively. The deduced amino acid sequence of each mature subunit showed high similarity with its counterparts in other teleost. Sequence analysis showed that npFSH-ß is more similar to higher vertebrate FSH-ßs than to higher vertebrate LH-ßs. Heterologous immunoassay was calibrated to analyse pituitary LH levels. While the LH immunoassay showed parallelism of npLH with that of tilapia (ta), no parallelism for FSH was found. Levels of pituitary LH were higher in females at gonadal stages of vitellogenic oocytes, mature secondary oocytes and mature tertiary oocytes with migrating nucleus than in pre-vitellogenic oocytes and early and late perinucleolus oocytes. Using competitive steroid ELISA, variations in the levels of the steroid hormones 11-ketotestosterone (11-KT) in males and E2 in females were characterized in relation to month and reproductive index of Nile perch. Our findings show that in females, gonadosomatic index and plasma E2 were highly correlated (R2 = 0.699, n = 172) and peaked from September to November while in males, the gonadosomatic index and plasma 11-KT peaked from October to November. In female fish, both steroid hormones were detected in the plasma but greatly varied in concentrations. E2 in particular, increased with the developmental stage of the gonads. The levels of steroid hormones, E2 and 11-KT in females and males respectively increased with fish size (total lengths) and suggest that females mature at a body length of 40-59 cm than their counter part males that mature at a total length of 60-70 cm. Taken together, we describe seasonal endocrine differences in wild-caught adult Nile perch which could potentially be exploited to manipulate the reproductive axis in cultured breeders.

Presence of GnRH3 in sturgeon and the roles of GnRH1 and GnRH2 on the regulation of LH/FSH in mature female sterlet Acipenser ruthenus in vitro.

Three forms of gonadotropin-releasing hormones (GnRHs), ArGnRH1, ArGnRH2, and ArGnRH3, were identified in sterlet. Compared with their orthologue, ArGnRH1 and ArGnRH2 have conserved core decapeptide but show low identity in the signal peptide and the rest of the sequences. The existence of the GnRH3 paralogue of sturgeon was predicted for the first time with TBLASTN by using the amino acid sequences of catshark and whale shark GnRH3 precursor as queries against the whole genome and transcript data of sterlet. The predicted ArGnRH3 cDNA sequence was composed of three exons containing all the elements of the GnRH family. The successful molecular cloning of GnRH3 from sterlets verified its expression in the brain of sturgeons. The analysis of the ArGnRH3 amino acid sequence revealed a completely conserved decapeptide sequence that shows 100% identity with the sequence of teleosts and differs in one amino acid with that of the cartilaginous fish (catshark and whale shark) at the 5th position. The structure of the phylogenetic tree showed that a total of 52 vertebrate GnRH sequences were clustered into three main clades corresponding to GnRH1, GnRH2, and GnRH3. The ArGnRH3 sequence is the oldest GnRH3 identified in teleosts. The tissue distribution analysis showed that ArGnRH1 was expressed in all the 13 examined tissues of females and in most of the tested tissues of male fish, with the highest expression in the pituitary and hypothalamus. ArGnRH2 is only expressed in the pituitary, hypothalamus, and gonads of both female and male sterlets. ArGnRH3 mRNA could be detected in the pituitary, hypothalamus, and gonad in both female and male fish. It is also present in the spleen, head kidney, and gill in female fish and in kidney and heart in male fish. However, the ArGnRH3 only showed weak expression in all the positive tissues. ArGnRH1 and ArGnRH2 active decapeptides were synthesized to investigate their roles on the regulation of LH/FSH using a mixed brain cell line from a sexually mature female sterlet. The results showed that ArGnRH1 and ArGnRH2 exerted different effects on the gene expression and release of gonadotropins. ArGnRH1 promoted the expression of fshß significantly around 48 h, and the expression was suppressed when the treatment time was extended to 72 h. ArGnRH1 had no significant effects on the level of either mRNA or secreted lh in any of the tested treatment length or concentrations. Moreover, ArGnRH1 did not stimulate the activity of gonadotropins in the maturation stage of female sturgeons. ArGnRH2 promoted the expression of fshß at 24 h and 48 h and increased mRNA level of lhß at 6 h and 48 h, accompanied by the significant secretion of LH at 72 h, although the high mRNA level of fsh did not correlate with the secretion of FSH in ArGnRH2-treated groups. In conclusion, ArGnRH2 plays an important role in the maturation stage of female sterlets. Therefore, ArGnRH2 has the potential to induce ovulation and spermiation in sturgeons.

The base pair-scale diffusion of nucleosomes modulates binding of transcription factors.

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone ß subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF's binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

Mutual Effects of Orexin and Bone Morphogenetic Proteins on Gonadotropin Expression by Mouse Gonadotrope Cells.

Orexin plays a key role in the regulation of sleep and wakefulness and in feeding behavior in the central nervous system, but its receptors are expressed in various peripheral tissues including endocrine tissues. In the present study, we elucidated the effects of orexin on pituitary gonadotropin regulation by focusing on the functional involvement of bone morphogenetic proteins (BMPs) and clock genes using mouse gonadotrope LßT2 cells that express orexin type 1 (OX1R) and type 2 (OX2R) receptors. Treatments with orexin A enhanced LHß and FSHß mRNA expression in a dose-dependent manner in the absence of GnRH, whereas orexin A in turn suppressed GnRH-induced gonadotropin expression in LßT2 cells. Orexin A downregulated GnRH receptor expression, while GnRH enhanced OX1R and OX2R mRNA expression. Treatments with orexin A as well as GnRH increased the mRNA levels of Bmal1 and Clock, which are oscillational regulators for gonadotropin expression. Of note, treatments with BMP-6 and -15 enhanced OX1R and OX2R mRNA expression with upregulation of clock gene expression. On the other hand, orexin A enhanced BMP receptor signaling of Smad1/5/9 phosphorylation through upregulation of ALK-2/BMPRII among the BMP receptors expressed in LßT2 cells. Collectively, the results indicate that orexin regulates gonadotropin expression via clock gene expression by mutually interacting with GnRH action and the pituitary BMP system in gonadotrope cells.

Effect of short- and long-term melatonin treatments on the reproductive activity of the tropical damselfish Chrysiptera cyanea.

Photoperiod plays a role in controlling the initiation and termination of reproduction in fish. Melatonin is an internal transducer of environmental photoperiod and is involved in regulating reproduction. The present study aimed to examine how melatonin impacts the transcript levels of kisspeptin (kiss1 and kiss2), gonadotropin-releasing hormones (gnrh1), and the ß-subunit of gonadotropins (fshß and lhß) in the brain of the sapphire devil, a tropical damselfish with long photoperiod preference. Feeding mature females with melatonin-containing pellets inhibited increases in the transcript levels of kiss1, gnrh1, and lhß within 3 h. Continuous melatonin treatment for 1 week resulted in oocyte regression and downregulation of kiss2, gnrh1, fshß, and lhß. When the transcript levels of kiss1 and gnrh1 were measured at 4-h intervals in the brain of sapphire devil, a day-high/night-low fluctuation was observed. The hypothalamic-pituitary-gonadal (HPG) axis may be influenced by melatonin, exerting a negative effect at night because the transcript levels of aralkylamine N-acetyltransferase (aanat2) increased during the scotophase. The expression of aanat2 was higher under short-day than long-day conditions, suggesting that there is a seasonal change in melatonin levels at night. It was concluded that change in photoperiod becomes a key factor for controlling the hormone synthesis in the HPG axis through melatonin.

Expression of genes that regulate follicle development and maturation during ovarian stimulation in poor responders.

RESEARCH QUESTION: Sex hormone-binding globulin (SHBG), androgen receptor (AR), LH beta polypeptide (LHB), progesterone receptor membrane component 1 (PGRMC1) and progesterone receptor membrane component 2 (PGRMC2) regulate follicle development and maturation. Their mRNA expression was assessed in peripheral blood mononuclear cells (PBMC) of normal and poor responders, during ovarian stimulation. DESIGN: Fifty-two normal responders and 15 poor responders according to the Bologna criteria were enrolled for IVF and intracytoplasmic sperm injection and stimulated with 200 IU of follitrophin alpha and gonadotrophin-releasing hormone antagonist. HCG was administered for final oocyte maturation. On days 1, 6 and 10 of stimulation, blood samples were obtained, serum hormone levels were measured, RNA was extracted from PBMC and real-time polymerase chain reaction was carried out to identify the mRNA levels. Relative mRNA expression of each gene was calculated by the comparative 2-DDCt method. RESULTS: Differences between mRNA levels of each gene on the same time point between the two groups were not significant. PGRMC1 and PGRMC2 mRNA levels were downregulated, adjusted for ovarian response and age. Positive correlations between PGRMC1 and AR (standardized beta = 0.890, P < 0.001) from day 1 to 6 and PGRMC1 and LHB (standardized beta = 0.806, P < 0.001) from day 1 to 10 were found in poor responders. PGRMC1 and PGRMC2 were positively correlated on days 6 and 10 in normal responders. CONCLUSIONS: PGRMC1 and PGRMC2 mRNA are significantly decreased during ovarian stimulation, with some potential differences between normal and poor responders.

Di-n-butyl phthalate diminishes testicular steroidogenesis by blocking the hypothalamic-pituitary-testicular axis: relationship with germ cell apoptosis in Japanese quail.

Although di-n-butyl phthalate (DBP) induces germ cell apoptosis, the underlying mechanism is not yet clear in quail. In this study, prepubertal quails were given a single dose of 500mg kg-1 DBP by gavage and were then killed 3, 6 and 24h after treatment. There was a significant reduction in intratesticular testosterone (ITT) concentrations and testicular steroidogenic enzyme mRNA expression and a significant increase in germ cell apoptosis in DBP-treated compared with control quails at all time points. Maximum apoptosis was detected 6h after treatment and the maximum reduction in testosterone concentrations was at 3h. To investigate whether DBP suppressed testicular steroidogenesis by affecting the hypothalamic-pituitary-testicular axis, we analysed pituitary LH subunit ß (Lhb) mRNA expression and serum LH concentrations. At all time points, pituitary Lhb expression and serum LH concentrations were significantly decreased following DBP treatment. The present observations suggest the possibility that DBP blocked LH secretion from the hypothalamus and/or pituitary, thereby decreasing LH stimulation of Leydig cells and reducing ITT concentrations. DBP-induced decreases in ITT concentrations may cause changes to the physical structure of Sertoli cells, which, in turn, may induce germ cell apoptosis.

Organization of the gonadotropin-inhibitory hormone (Lpxrfa) system in the brain of zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. However, the role of GnIH (Lpxrfa) in teleosts is unknown. In this study, a transgenic zebrafish (Danio rerio) line Tg(gnih:mCherry) was developed to determine the organization of GnIH neurons in the brain. Another transgenic line, Tg(gnih:mCherry; gnrh3:eGFP), was established to determine the positional relationships between GnIH and GnRH3 neurons. In these transgenic lines, the mCherry protein was specifically expressed in GnIH neurons, and eGFP was expressed exclusively in GnRH3 neurons. We found that GnIH cell somata were restricted to the posterior periventricular nucleus (NPPv). Most GnIH neuronal processes projected to the hypothalamus, but a few extended to the posterior tuberculum, telencephalon, and olfactory bulb. GnIH neuronal processes were in close apposition with GnRH3 cell somata and processes in the preoptic-hypothalamic area but were seldom in direct contact. However, in the olfactory bulb, GnIH neuronal processes were in proximity to the terminal nerve GnRH3 cell somata. Neither GnIH cell soma nor neuronal processes were detected in the pituitary, although GnIH receptor mRNAs (npffr1l1, npffr1l2, and npffr1l3) were detected. Intraperitoneal administration of GnIH-3 peptides promoted the transcription of brain gnrh3 as well as pituitary fshß but not lhß. Thus, GnIH cell somata were specifically distributed in the NPPv, and their fibers extended to the hypothalamus and advanced to the telencephalon and olfactory bulb. We conclude that GnIH may directly stimulate terminal nerve GnRH3 neurons in the zebrafish brain.

Effect of anti-Müllerian hormone on the regulation of pituitary gonadotropin subunit expression: roles of kisspeptin and its receptors in gonadotroph LßT2 cells.

Anti-Müllerian hormone (AMH) is primarily produced by ovarian granulosa cells and contributes to follicle development. AMH is also produced in other tissues, including the brain and pituitary; however, its roles in these tissues are not well understood. In this study, we examined the effect of AMH on pituitary gonadotrophs. We detected AMH and AMH receptor type 2 expression in LßT2 cells. In these cells, the expression of FSHß- but not α- and LHß-subunits increased significantly as the concentration of AMH increased. LßT2 cells expressed Kiss-1 and Kiss-1R. AMH stimulation resulted in decreases in both Kiss-1 and Kiss-1R. The siRNA-mediated knockdown of Kiss-1 in LßT2 cells did not alter the basal expression levels of α-, LHß-, and FSHß-subunits. In LßT2 cells overexpressing Kiss-1R, exogenous kisspeptin stimulation significantly increased the expression of all three gonadotropin subunits. However, kisspeptin-induced increases in these subunits were almost completely eliminated in the presence of AMH. In contrast, GnRH-induced increases in the three gonadotropin subunits were not modulated by AMH. Our observations suggested that AMH acts on pituitary gonadotrophs and induces FSHß-subunit expression with concomitant decreases in Kiss-1 and Kiss-1R gene expression. Kisspeptin, but not GnRH-induced gonadotropin subunit expression, was inhibited by AMH, suggesting that it functions in association with the kisspeptin/Kiss-1R system in gonadotrophs.

The Genetic Backdrop of Hypogonadotropic Hypogonadism.

The pituitary is an organ of dual provenance: the anterior lobe is epithelial in origin, whereas the posterior lobe derives from the neural ectoderm. The pituitary gland is a pivotal element of the axis regulating reproductive function in mammals. It collects signals from the hypothalamus, and by secreting gonadotropins (FSH and LH) it stimulates the ovary into cyclic activity resulting in a menstrual cycle and in ovulation. Pituitary organogenesis is comprised of three main stages controlled by different signaling molecules: first, the initiation of pituitary organogenesis and subsequent formation of Rathke's pouch; second, the migration of Rathke's pouch cells and their proliferation; and third, lineage determination and cellular differentiation. Any disruption of this sequence, e.g., gene mutation, can lead to numerous developmental disorders. Gene mutations contributing to disordered pituitary development can themselves be classified: mutations affecting transcriptional determinants of pituitary development, mutations related to gonadotropin deficiency, mutations concerning the beta subunit of FSH and LH, and mutations in the DAX-1 gene as a cause of adrenal hypoplasia and disturbed responsiveness of the pituitary to GnRH. All these mutations lead to disruption in the hypothalamic-pituitary-ovarian axis and contribute to the development of primary amenorrhea.

The effects of luteinising hormone gene polymorphism on the outcomes of in vitro fertilisation and embryo transfer.

Trp8Arg polymorphism of the LH beta gene has decreased bioactivity in vivo and previous studies showed conflicting data on the effect of LH beta gene polymorphism on the IVF outcome. In this study, 591 IVF patients were recruited. Patients with the variant allele(s) were the carrier group. In GnRH antagonist cycles, the clinical pregnancy rate was significantly lower in the carrier group (18.9%) than in the noncarrier group (37.1%). In long GnRH agonist cycles, the clinical pregnancy rate was comparable between both groups. To clarify the effect of COH protocols, IVF outcomes in the GnRH antagonist and long GnRH agonist protocol groups in carriers were analysed. Among carriers, the clinical pregnancy rate was significantly lower in the GnRH antagonist protocol group (18.9%) than in the long GnRH agonist protocol group (45.2%). Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future.Impact StatementWhat is already known on this subject? Trp8Arg polymorphism of the LH beta gene is known to have decreased bioactivity in vivo. Previous studies have demonstrated hypo-sensitivity in the patients with the variant LH beta protein, while other study showed similar carrier frequency between the poor and the normal response group.What the results of this study add? The variant LH beta gene was associated with a lower clinical pregnancy rate in GnRH antagonist cycles but not in long GnRH agonist cycles.What the implications are of these findings for clinical practice and/or further research? Single nucleotide polymorphism analysis may contribute to the individualisation of COH protocols for each patient in the future.

Effect of dietary pomegranate peel powder on productive traits, blood chemistry, economic efficiency and the expression of FSHR and LH-ß genes in two strains of laying Japanese quail.

The current experiment was designed to study the response of two strains of laying Japanese quail to diet containing different levels (0%, 3%, 6%, and 9%) of pomegranate peel (PP) powder for a period of 6 weeks, on egg production, blood chemistry, carcass traits, expression of FSHR and LH-ß genes, and economic efficiency. A total of 576 6-week-old Japanese quails were made up of two strains: white quails (n=288) and brown quails (n=288). Four treatment groups were randomly distributed for each strain. Each group was subdivided into 6 replicates of 10 birds each. Results revealed that the white strain showed significant (P<0.05) higher values in final body weight (BW), egg quality parameters, weights of dressed carcass, and total return (TR) compared with the brown strain. Brown strain had higher hen day egg production % (HDEP%). A significant (P<0.05) interaction effects between genetic type and the dietary PP powder levels was recorded in growth traits, some egg production traits, and mRNA expression of FSHR and LH-ß genes. Results concluded that all the dietary levels of PP powder up to 9% improved growth traits, egg production traits, fertility% (P=0.001), and hatchability% (P=0.007). Moreover, they have a safely biochemical effect on the level of urea (P=0.002) and the concentration of aspartate amino transferase (AST) (P<0.001). It also enhanced mRNA expression of FSHR and LH-ß genes, but showed non-significant (P>0.05) influence on carcass traits. Economically, the net return (NR) was increased in quails fed on 6% and 9% PP powder diets.

Short-term but not long-term high-fat diet induces an increase in gene expression of gonadotropic hormones and GPR120 in the male mouse pituitary gland.

High-fat diet (HFD) is associated with the regulation of reproductive functions. This study aimed to investigate the effects of short-term HFD on the mRNA expression levels of follicle-stimulating hormone ß subunit (FSHß), luteinizing hormone ß subunit (LHß), gonadotropin-releasing hormone receptor, and long-chain fatty acid receptor, GPR120, in the matured male mouse pituitary gland. Adult male mice were fed either control chow or HFD for 1, 2, 5, 10, 30 and 150 days. Fshb and Gpr120 mRNA expression levels in the pituitary glands were significantly increased during 2 to 30 days of HFD feeding. Gnrh-r mRNA in the 30 days HFD fed group and body weight in the 30 and 150 days HFD fed groups were higher than control. However, there were no significant differences in plasma non-esterified fatty acids or glucose levels during the 150 days of HFD feeding. These results suggest that male mice feeding a short-term HFD induces FSHß synthesis and GPR120 expression in their pituitary gonadotropes.

Colocalization of GPR120 and anterior pituitary hormone-producing cells in female Japanese Black cattle.

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone ß subunit (LHß)- and follicle-stimulating hormone ß subunit (FSHß)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

AMP-activated protein kinase activation reduces the transcriptional activity of the murine luteinizing hormone ß-subunit gene.

Malnutrition is one of the factors that induces reproductive disorders. However, the underlying biological processes are unclear. AMP-activated protein kinase (AMPK) is an enzyme that plays crucial role as a cellular energy sensor. In the present study, we examined the effects of AMPK activation on the transcription of the murine gonadotropin subunit genes Cga, Lhb, and Fshb, and the gonadotropin-releasing hormone receptor Gnrh-r. Real-time PCR and transcription assay using LßT2 cells demonstrated that 5-amino-imidazole carboxamide riboside (AICAR), a cell-permeable AMP analog, repressed the expression of Lhb. Next, we examined deletion mutants of the upstream region of Lhb and found that the upstream regulatory region of Lhb (-2527 to -2198 b) was responsible for the repression by AICAR. Furthermore, putative transcription factors (SP1, STAT5a, and TEF) that might mediate transcriptional control of the Lhb repression induced by AICAR were identified. In addition, it was confirmed that both AICAR and a competitive inhibitor of glucose metabolism, 2-deoxy-D-glucose, induced AMPK phosphorylation in LßT2 cells. Therefore, the upstream region of Lhb is one of the target sites for glucoprivation inducing AMPK activation. In addition, AMPK plays a role in repressing Lhb expression through the distal -2527 to -2198 b region.

Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids.

GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LßT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LßT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LßT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.

The mRNA expression patterns of kisspeptins, GnRHs, and gonadotropins in the brain and pituitary gland of a tropical damselfish, Chrysiptera cyanea, during the reproductive cycle.

The sapphire devil (Chrysiptera cyanea) is a tropical damselfish that undergoes active reproduction under long-day conditions. To elucidate the physiological regulation of the brain-pituitary-gonadal axis in female sapphire devil, we cloned and characterized the genes of two kisspeptins (kiss1 and kiss2), three gonadotropin-releasing hormones (gnrh1, gnrh2, gnrh3), and the ß-subunit of two gonadotropins (fshß and lhß) and investigated the gene expression changes during ovarian development. Quantitative polymerase chain reaction analyses in various brain parts revealed high expression levels of kiss1, kiss2, and gnrh2 in the diencephalon; gnrh2 and gnrh3 in the telencephalon; and fshß and lhß in the pituitary. In situ hybridization (ISH) analyses revealed positive signals of kiss1 in the dorsal and ventral habenular nucleus and of kiss2 in the dorsal and ventral parts of the nucleus of the lateral recess. This analysis showed gnrh1 expression in the preoptic area (POA), suggesting that GnRH1 plays a stimulating role in the secretion of gonadotropins from the pituitary of the sapphire devil. High transcription levels of kiss1, kiss2, gnrh1, gnrh2, fshß, and lhß were observed in the brain during the late vitellogenic stage, suggesting their involvement in the physiological processes of vitellogenesis. Immersion of fish in estradiol-17ß (E2)-containing seawater resulted in increased expression of kiss2 and gnrh1 in their brains. This study showed that kiss-expressing neurons in the diencephalon are influenced by E2, leading to upregulation of gnrh1 in the POA and of fshß and lhß in the pituitary during vitellogenesis.