Studies using lipid infusions to raise fatty acid levels require heparin to release lipoprotein lipase (LPL), thus calling into question the appropriate control infusion for this type of study: saline alone or saline plus heparin. We aimed to evaluate whether the addition of heparin alone, in doses needed to release LPL, would alter circulating free fatty acids (FFAs) and/or affect gonadotropins.

Time-resolved fluoroimmunoassay (TR-FIA), which has recently been developed as a non-isotopic immunoassay, was intended for assay of gonadotropins (LH and FSH). Ovine LH or FSH was labeled with N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2, N3, N3-tetraacetic acid Eu-chelate (DTTA), and competitive binding assay was performed using a 96-weel titer plate previously coated with a second antibody, followed by measurement using time-resolved fluorometry. TR-FIA for standard ovine LH (NIDDK, LH-I-3) or FSH (NIDDK, FSH-I-1) had a sensitivity of about 25 pg/50 microliters sample. The assay system was applied to heterologous assay of porcine gonadotropins. Linearity was obtained by the dilution test using medium from primary culture of porcine anterior pituitary cells. Intra- and inter-assay coefficients of variation of LH and FSH determination in 31 different porcine samples were satisfactorily low, between 3.5 and 8.1% (intra-assay) and between 1.7 and 13.1% (inter-assay). Correlation coefficients between radioimmunoassay and TR-FIA were calculated to be 0.945 for LH and 0.978 for FSH. Stimulation of LH and FSH release with GnRH was observed by TR-FIA. This non-isotopic TR-FIA thus provides as good sensitivity, reproducibility and accuracy as conventional RIAs.

The gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are important hormones in vertebrate reproduction. The isolation of gonadotropins from the pituitary gland is sub-optimal, as the cross-contamination of one hormone with another is common and often results in the variation in the measured activity of LH and FSH. The production of recombinant hormones is, therefore, a viable approach to solve this problem. This study aimed to express recombinant rat, mouse, and mastomys FSH and LH in Chinese hamster ovary (CHO) cells. Their common α-subunits along with their hormone-specific β-subunits were encoded in a single mammalian expression vector. FSH from all three species was expressed, whereas expression was achieved only for the mouse LH. Immunohistochemistry for rat alpha subunit of glycoprotein hormone (αGSU) and LHβ and FSHβ subunits confirmed the production of the dimeric hormone in CHO cells. The recombinant rodent gonadotropins were confirmed to be biologically active; estradiol production was increased by recombinant FSH in granulosa cells, while recombinant LH increased testosterone production in Leydig cells.

In fish, both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play important roles in reproduction. Here we explored the structure and differential specificity of tilapia (t) gonadotropins (GTHs) to delineate their physiological relevance and the nature of their regulation. We generated structural models of tGTHs and GTH receptors (R) that enabled us to better understand the hormone-receptor interacting region. In tilapia, FSH release is under the control of the hypothalamic decapeptide GnRH, an effect that was abolished by specific bioneutralizing antisera [anti-recombinant (r) tFSHβ]. These antisera also reduced the basal secretion and delayed GnRH-stimulated production of 11-ketotestosterone (11KT), and dramatically reduced LH levels. Immunoneutralization of tLH using anti-rtLHβ significantly reduced its GnRH-stimulated levels. Basal 11KT and FSH levels were also reduced. Taken together, these results suggest a feedback mechanism between FSH and LH release in tilapia.

The synthesis and secretion of the gonadotropic hormones involves coordination of signal transduction, gene expression, protein translation, post-translational folding and modification and finally secretion. The production of biologically active gonadotropin thus requires appropriately folded and glycosylated subunits that assemble to form the heterodimeric hormone. Here we overview recent literature on regulation of gonadotropin subunit gene expression and current understanding of the assembly and secretion of biologically active gonadotropic hormones. Finally, we discuss the therapeutic potential of understanding glycosylation function towards designing new forms of gonadotropins based on observations of physiologically relevant parameters such as age related glycosylation changes.

Gonadotropins can be administered every 5 days under intradermal injection in in vitro fertilization (IVF) treatment.

Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. This report examined the expression of Prx isotype I in the rat ovary after hormone treatment.

Gonadotropins are required for gametogenesis but in embryonic gonads this mechanism is not well understood. Here we use chicken embryos to investigate the mechanism that gonadotropins regulate the ovarian germ cell mitosis/meiosis decision. Treatment with follicle-stimulating hormone (FSH) delayed germ cell meiosis entry and promoted their proliferation. This action was blocked by an aromatase inhibitor. Treatment with luteinizing hormone (LH) accelerated germ cell meiosis entry and promoted transcription of 3βHSDII to increase progesterone (P4) production. In the cultured ovaries, P4 triggered meiotic initiation in germ cells. MiR181a, which acts to downregulate the NR6A1 transcript to inhibit the meiotic initiation, was upregulated by FSH and downregulated by LH. Collectively, gonadotropins regulate germ cells mitosis and meiotic initiation through steroid hormones and a miR181a-mediated pathway. In particularly, FSH delays germ cell meiosis entry and promotes cell proliferation via estrogen while LH accelerates the meiotic initiation via elevated P4 production.

Pituitary gonadotropins follicle-stimulating hormone and luteinizing hormone are heterodimeric glycoproteins expressed in gonadotropes. They act on gonads and promote their development and functions including steroidogenesis and gametogenesis. Although transcriptional regulation of gonadotropin subunits has been well studied, the post-transcriptional regulation of gonadotropin subunits is not well understood. To test if microRNAs regulate the hormone-specific gonadotropin β subunits in vivo, we deleted Dicer in gonadotropes by a Cre-lox genetic approach. We found that many of the DICER-dependent microRNAs, predicted in silico to bind gonadotropin β subunit mRNAs, were suppressed in purified gonadotropes of mutant mice. Loss of DICER-dependent microRNAs in gonadotropes resulted in profound suppression of gonadotropin-β subunit proteins and, consequently, the heterodimeric hormone secretion. In addition to suppression of basal levels, interestingly, the post-gonadectomy-induced rise in pituitary gonadotropin synthesis and secretion were both abolished in mutants, indicating a defective gonadal negative feedback control. Furthermore, mutants lacking Dicer in gonadotropes displayed severely reduced fertility and were rescued with exogenous hormones confirming that the fertility defects were secondary to suppressed gonadotropins. Our studies reveal that DICER-dependent microRNAs are essential for gonadotropin homeostasis and fertility in mice. Our studies also implicate microRNAs in gonadal feedback control of gonadotropin synthesis and secretion. Thus, DICER-dependent microRNAs confer a new layer of transcriptional and post-transcriptional regulation in gonadotropes to orchestrate the hypothalamus-pituitary-gonadal axis physiology.

A preponderance of basic luteinizing hormone (LH) molecules having elevated bioactivity was detected in the circulation of women with polycystic ovary syndrome (PCOS). Subsequent studies have shown that LH and follicle-stimulating hormone (FSH) both circulate as glycoforms differing in number of glycans: low-N-glycosylated glycoforms, LHdi and FSHtri, with high in vitro bioactivity, and fully glycosylated glycoforms, LHtri and FSHtetra, with high in vivo bioactivity.

Hypogonadotropic hypogonadism is characterized by inadequate secretion of pituitary gonadotropins, leading to absent, partial, or arrested puberty. In males, classical treatment with testosterone promotes virilization but not testicular growth or spermatogenesis. To quantify treatment practices and efficacy, we systematically reviewed all studies investigating gonadotropins for the achievement of pubertal outcomes in males with hypogonadotropic hypogonadism.

Nandrolone decanoate (ND) is an anabolic androgenic steroid (AAS) which influences the ovarian structure and function. We assessed the effects of ND on the ovarian volume, number of primordial follicles, and level of hormones and also evaluated the modulatory effects of gonadotropins on the histopathological changes imposed by the administration of ND.

Female reproductive function depends on a choreographed sequence of hormonal secretion and action, where specific stresses such as inflammation exert profound disruptions. Specifically, acute LPS-induced inflammation inhibits gonadotropin production and secretion from the pituitary, thereby impacting the downstream production of sex hormones. These outcomes have only been observed in acute inflammatory stress and little is known about the mechanisms by which chronic inflammation affects reproduction. In this study we seek to understand the chronic effects of LPS on pituitary function and consequent luteinizing and follicle stimulating hormone secretion.

Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective β subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHβ or cFSHβ mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH β-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHβ or rcFSHβ, respectively, and rcLHβα or rcFSHβα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species - the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels - but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.

Echinomycin is a small-molecule inhibitor of hypoxia- inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1α mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1α and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1α and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1α directly mediated the transcriptional activation of ET-2 during gonadotropin- induced superuvulation. Taken together, these results demonstrated that HIF-1α-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.

The gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and their receptors play critical roles in vertebrate reproduction. In order to study intra- and interspecies ligand promiscuity of gonadotropins, COS-7 cells were transiently transfected with one of the gonadotropin receptor genes, FSHR or LHR, and tested for activation by gonadotropins from representative fish orders: Aquilliformes (eel; e), Salmoniformes (trout; tr), and Perciformes (tilapia; ta), and of mammalian origin: porcine (p), bovine (b) and human (h). The study reveals complex relations between the gonadotropin hormones and their receptors. Each gonadotropin activated its own cognate receptor. However, taLHR was also activated by hCG and eLHR was activated by hFSH, hCG, and trFSH. For FSHR, the only cross-reactivity detected was for hFSHR, which was activated by pFSH and bFSH. These findings are of great interest and applicability in the context of activation of various GTHRs by their ligands and by ligands from other vertebrates. Analysis of the three-dimensional models of the structures highlights the importance of residues outside of the currently established hormone-receptor interface region. In addition, the interface residues in taFSHR and the effect of exon duplication, which causes an insert in the LRR domain, are suggested to affect the interaction and binding of taFSH.

Ovarian follicular development is primarily dependent on pituitary gonadotropins. Identification of gonadotropin-inducible genes in the ovary is one of the effective approaches for the study of follicular development. In this study we identify rat homologue of p120, a nuclear transcription co-activator, as one of the FSH inducible genes in the rat granulosa cells.

Colorectal cancer (CRC) is a leading cause of death in the western world, and its incidence increases with patient age. It is also known that with age there occur changes in the levels of certain hormones, including an increase in the secretion of pituitary gonadotropins (PtGs) as a result of the loss of gonadal hormone feedback. We recently reported that functional PtG receptors are expressed in human lung cancer cells, rhabdomyosarcoma cells, and malignant hematopoietic stem cells.

Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein TEpacVV in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.