Unconventional Actions of Glycoprotein Hormone Subunits: A Comprehensive Review.

The glycoprotein hormones (GPH) are heterodimers composed of a common α subunit and a specific ß subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The α subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin ß subunit (ßhCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) ß subunit is also reported to have biological effects. The GPH α- and ß subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.

Origin and Evolution of the Neuroendocrine Control of Reproduction in Vertebrates, With Special Focus on Genome and Gene Duplications.

In humans, as in the other mammals, the neuroendocrine control of reproduction is ensured by the brain-pituitary gonadotropic axis. Multiple internal and environmental cues are integrated via brain neuronal networks, ultimately leading to the modulation of the activity of gonadotropin-releasing hormone (GnRH) neurons. The decapeptide GnRH is released into the hypothalamic-hypophysial portal blood system and stimulates the production of pituitary glycoprotein hormones, the two gonadotropins luteinizing hormone and follicle-stimulating hormone. A novel actor, the neuropeptide kisspeptin, acting upstream of GnRH, has attracted increasing attention in recent years. Other neuropeptides, such as gonadotropin-inhibiting hormone/RF-amide related peptide, and other members of the RF-amide peptide superfamily, as well as various nonpeptidic neuromediators such as dopamine and serotonin also provide a large panel of stimulatory or inhibitory regulators. This paper addresses the origin and evolution of the vertebrate gonadotropic axis. Brain-pituitary neuroendocrine axes are typical of vertebrates, the pituitary gland, mediator and amplifier of brain control on peripheral organs, being a vertebrate innovation. The paper reviews, from molecular and functional perspectives, the evolution across vertebrate radiation of some key actors of the vertebrate neuroendocrine control of reproduction and traces back their origin along the vertebrate lineage and in other metazoa before the emergence of vertebrates. A focus is given on how gene duplications, resulting from either local events or from whole genome duplication events, and followed by paralogous gene loss or conservation, might have shaped the evolutionary scenarios of current families of key actors of the gonadotropic axis.

Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.

BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood. RESULTS: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription. CONCLUSION: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.

A regulatory loop between miR-132 and miR-125b involved in gonadotrope cells desensitization to GnRH.

The GnRH neurohormone is the main activator of the pituitary gonadotropins, LH and FSH. Here we investigated the contribution of microRNAs in mediating GnRH activation. We first established that miR-125b targets several actors of Gαq/11 signalling pathway, without altering Gαs pathway. We then showed that a Gαs-mediated, PKA-dependent phosphorylation of NSun2 methyltransferase leads to miR-125b methylation and thereby induces its down-regulation. We demonstrated that NSun2 mRNA is a target of miR-132 and that NSun2 may be inactivated by the PP1α phosphatase. Time-course analysis of GnRH treatment revealed an initial NSun2-dependent down-regulation of miR-125b with consecutive up-regulation of LH and FSH expression. Increase of miR-132 and of the catalytic subunit of PP1α then contributed to NSun2 inactivation and to the return of miR-125b to its steady-state level. The Gαq/11-dependent pathway was thus again silenced, provoking the down-regulation of LH, FSH and miR-132. Overall, this study reveals that a regulatory loop that tends to maintain or restore high and low levels of miR-125b and miR-132, respectively, is responsible for gonadotrope cells desensitization to sustained GnRH. A dysregulation of this loop might be responsible for the inverted dynamics of these two miRNAs reported in several neuronal and non-neuronal pathologies.

Epigenetic regulation of alternative promoters and enhancers in progenitor, immature, and mature gonadotrope cell lines.

Gonadotrope cell identity genes emerge in a stepwise process during mouse pituitary development. Cga, encoding for the α-subunit of TSH, LH, and FSH, is initially detected at E11.5 followed by Gnrhr and steroidogenic factor Sf1 at E13.5, specifying cells engaged in a gonadotrope cell fate. Lhb and Fshb appear at E16.5 and 17.5, respectively, typifying differentiated gonadotrope cells. Using the αT1-1, αT3-1 and LßT2 cell lines recapitulating these stages of gonadotrope differentiation, DNA methylation at Gnrhr and Sf1 was investigated. Regulatory regions were found hypermethylated in progenitor αT1-1 cells and hypomethylated in differentiated LßT2 cells. Abundance of RNA polymerase II together with active histone modifications including H3K4me1, H3K4me3, and H3K27ac were strictly correlated with DNA hypomethylation. Analyses of epigenomic modifications and chromatin accessibility were further extended to Isl1, Lhx3, Gata2, and Pitx2, highlighting alternative usages of specific regulatory gene domains in progenitor αT1-1, immature αT3-1, and mature LßT2 gonadotrope cells.

Rapid communication: A microRNA-132/212 pathway mediates GnRH activation of FSH expression.

GnRH plays a key role in the vertebrate reproductive system by stimulating biosynthesis and secretion of pituitary gonadotropins. However, the potential involvement of microRNAs (miRNAs) on this activation has still to be explored. In this study, we investigated the role of miRNA-132 and miRNA-212, two tandemly expressed miRNAs that target the same transcripts, on GnRH-induced FSH expression. We first showed that the GnRH stimulation of FSH secretion was reduced and Fshb mRNA abolished by blocking miR-132/212 action in rat pituitary cells. In mouse LßT2 gonadotrope cells, the GnRH stimulation of Fshb mRNA was also demonstrated to be dependent on miR-132/212 and reproduced by overexpressing one or both miRNAs. We then showed that the miR-132/212-mediated action of GnRH involved a posttranscriptional decrease of sirtuin 1 (SIRT1) deacetylase. The lower level of SIRT1 deacetylase correlated with an increase in the acetylated form of Forkhead Box O1 (FOXO1), a transcriptional repressor of Fshb. Interestingly, we show that the acetylated mimicking mutant of FOXO1 was localized outside the nucleus, thus alleviating its repressive effect on Fshb transcription. Overall, we demonstrate that the GnRH stimulation of Fshb expression is dependent on miR-132/212 and involves a SIRT1-FOXO1 pathway. This is the first demonstration of an obligatory microRNA pathway in the GnRH-regulated expression of a gonadotropin gene.

Multiple thyrotropin ß-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

Thyroid-stimulating hormone (TSH) is composed of a specific ß subunit and an α subunit that is shared with the two pituitary gonadotropins. The three ß subunits derive from a common ancestral gene through two genome duplications (1R and 2R) that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshß subunit-related gene that was generated through 2R. This gene, named Tshß2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr) gene in these species suggests that both TSHs act through the same receptor. A novel Tshß sister gene, named Tshß3, was generated through the third genomic duplication (3R) that occurred early in the teleost lineage. Tshß3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshßs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshß and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshß3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

Looking for the bird Kiss: evolutionary scenario in sauropsids.

BACKGROUND: The neuropeptide Kiss and its receptor KissR are key-actors in the brain control of reproduction in mammals, where they are responsible for the stimulation of the activity of GnRH neurones. Investigation in other vertebrates revealed up to 3 Kiss and 4 KissR paralogs, originating from the two rounds of whole genome duplication in early vertebrates. In contrast, the absence of Kiss and KissR has been suggested in birds, as no homologs of these genes could be found in current genomic databases. This study aims at addressing the question of the existence, from an evolutionary perspective, of the Kisspeptin system in birds. It provides the first large-scale investigation of the Kisspeptin system in the sauropsid lineage, including ophidian, chelonian, crocodilian, and avian lineages. RESULTS: Sauropsid Kiss and KissR genes were predicted from multiple genome and transcriptome databases by TBLASTN. Phylogenetic and syntenic analyses were performed to classify predicted sauropsid Kiss and KissR genes and to re-construct the evolutionary scenarios of both gene families across the sauropsid radiation.Genome search, phylogenetic and synteny analyses, demonstrated the presence of two Kiss genes (Kiss1 and Kiss2 types) and of two KissR genes (KissR1 and KissR4 types) in the sauropsid lineage. These four genes, also present in the mammalian lineage, would have been inherited from their common amniote ancestor. In contrast, synteny analyses supported that the other Kiss and KissR paralogs are missing in sauropsids as in mammals, indicating their absence in the amniote lineage. Among sauropsids, in the avian lineage, we demonstrated the existence of a Kiss2-like gene in three bird genomes. The divergence of these avian Kiss2-like sequences from those of other vertebrates, as well as their absence in the genomes of some other birds, revealed the processes of Kiss2 gene degeneration and loss in the avian lineage. CONCLUSION: These findings contribute to trace back the evolutionary history of the Kisspeptin system in amniotes and sauropsids, and provide the first molecular evidence of the existence and fate of a Kiss gene in birds.

Identification and analysis of two novel sites of rat GnRH receptor gene promoter activity: the pineal gland and retina.

BACKGROUND AND AIMS: In mammals, activation of pituitary GnRH receptor (GnRHR) by hypothalamic GnRH increases the synthesis and secretion of LH and FSH, which, in turn, regulate gonadal functions. However, GnRHR gene (Gnrhr) expression is not restricted to the pituitary. METHODS: To gain insight into the extrapituitary expression of Gnrhr, a transgenic mouse model that expresses the human placental alkaline phosphatase reporter gene driven by the rat Gnrhr promoter was created. RESULTS: This study shows that the rat Gnrhr promoter is operative in two functionally related organs, the pineal gland, as early as embryonic day (E) 13.5, and the retina where activity was only detected at E17.5. Accordingly, Gnrhr mRNA were present in both tissues. Transcription factors known to regulate Gnrhr promoter activity such as the LIM homeodomain factors LHX3 and ISL1 were also detected in the retina. Furthermore, transient transfection studies in CHO and gonadotrope cells revealed that OTX2, a major transcription factor in both pineal and retina cell differentiation, is able to activate the Gnrhr promoter together with either CREB or PROP1, depending on the cell context. CONCLUSION: Rather than using alternate promoters, Gnrhr expression is directed to diverse cell lineages through specific associations of transcription factors acting on distinct response elements along the same promoter. These data open new avenues regarding GnRH-mediated control of seasonal and circadian rhythms in reproductive physiology.

GATA2-induced silencing and LIM-homeodomain protein-induced activation are mediated by a bi-functional response element in the rat GnRH receptor gene.

GATA2 transcription factor and LIM homeodomain proteins Islet1 (ISL1) and LIM homeobox 3 (LHX3) are suspected to be involved in gonadotrope cell fate and maintenance. The GnRH receptor gene (Gnrhr), crucial for gonadotrope function, is expressed in the pituitary gland from embryonic day 13.5 onward, well before LH and FSH ß-subunits. This expression pattern together with the presence of WGATAR and TAAT motifs in Gnrhr promoter sequences suggests the involvement of early transcription factors in promoter activation. In this study, using a well-characterized transgenic mouse model, GATA2 was found colocalized with Gnrhr promoter activity in the pituitary. Transient transfection of Gnrhr promoter luciferase fusion constructs together with either GATA2 expression vectors or small interfering RNA in gonadotrope cell lines indicated that GATA2, which typically acts as a trans-activator, unexpectedly repressed Gnrhr promoter activity. Using DNA chromatography affinity and EMSA, we demonstrated that GATA2 operates via a response element containing a peculiar palindromic GATA motif that overlaps a critical TAAT motif involved in LHX3/ISL1 trans-activation. Indeed, despite the inhibitory action of GATA2, this element displayed a clear-cut enhancer activity in gonadotrope cells. Chromatin immunoprecipitation assays indicated that GATA2, LHX3, and ISL1 interact with a Gnrhr promoter fragment encompassing this element. The trans-repressive action of GATA2 on Gnrhr promoter activity is likely balanced or even hindered by trans-activating effects of LIM homeodomain proteins via this novel bifunctional LIM/GATA response element. Such a hierarchical interplay may contribute to finely adjust Gnrhr gene expression in gonadotrope cell lineage during pituitary development as well as in the adult animal.

Mechanisms underlying the tissue-specific and regulated activity of the Gnrhr promoter in mammals.

The GnRH receptor (GnRHR) plays a central role in the development and maintenance of reproductive function in mammals. Following stimulation by GnRH originating from the hypothalamus, GnRHR triggers multiple signaling events that ultimately stimulate the synthesis and the periodic release of the gonadotropins, luteinizing-stimulating hormone (LH) and follicle-stimulating hormones (FSH) which, in turn, regulate gonadal functions including steroidogenesis and gametogenesis. The concentration of GnRHR at the cell surface is essential for the amplitude and the specificity of gonadotrope responsiveness. The number of GnRHR is submitted to strong regulatory control during pituitary development, estrous cycle, pregnancy, lactation, or after gonadectomy. These modulations take place, at least in part, at the transcriptional level. To analyze this facet of the reproductive function, the 5' regulatory sequences of the gene encoding the GnRHR have been isolated and characterized through in vitro and in vivo approaches. This review summarizes results obtained with the mouse, rat, human, and ovine promoters either by transient transfection assays or by means of transgenic mice.

Emergence and evolution of the glycoprotein hormone and neurotrophin gene families in vertebrates.

BACKGROUND: The three vertebrate pituitary glycoprotein hormones (GPH) are heterodimers of a common α and a specific ß subunit. In human, they are located on different chromosomes but in a similar genomic environment. We took advantage of the availability of genomic and EST data from two cartilaginous fish species as well as from two lamprey species to identify their repertoire of neurotrophin, lin7 and KCNA gene family members which are in the close environment of gphß. Gphα and gphß are absent outside vertebrates but are related to two genes present in both protostomes and deuterostomes that were named gpa2 and gpb5. Genomic organization and functional characteristics of their protein products suggested that gphα and gphß might have been generated concomitantly by a duplication of gpa2 and gpb5 just prior to the radiation of vertebrates. To have a better insight into this process we used new genomic resources and tools to characterize the ancestral environment before the duplication occurred. RESULTS: An almost similar repertoire of genes was characterized in cartilaginous fishes as in tetrapods. Data in lampreys are either incomplete or the result of specific duplications and/or deletions but a scenario for the evolution of this genomic environment in vertebrates could be proposed. A number of genes were identified in the amphioxus genome that helped in reconstructing the ancestral environment of gpa2 and gpb5 and in describing the evolution of this environment in vertebrates. CONCLUSION: Our model suggests that vertebrate gphα and gphß were generated by a specific local duplication of the ancestral forms of gpa2 and gpb5, followed by a translocation of gphß to a new environment whereas gphα was retained in the gpa2-gpb5 locus. The two rounds of whole genome duplication that occurred early in the evolution of vertebrates generated four paralogues of each gene but secondary gene losses or lineage specific duplications together with genomic rearrangements have resulted in the present organization of these genes, which differs between vertebrate lineages.

Distinct expression patterns of glycoprotein hormone-alpha2 and -beta5 in a basal chordate suggest independent developmental functions.

The vertebrate glycoprotein hormones (GpHs), gonadotropins and thyrotropin, are heterodimers composed of a common alpha- and specific beta-subunit. The recombinant heterodimer of two additional, structurally related proteins identified in vertebrate and protostome genomes, the glycoproteins-alpha2 (GPA2) and-beta5 (GPB5), was shown to activate the thyrotropin receptor and was therefore named thyrostimulin. However, differences in tissue distribution and expression levels of these proteins suggested that they might act as nonassociated factors, prompting further investigation on these proteins. In this study we show that GPA2 and GPB5 appeared with the emergence of bilateria and were maintained in most groups. These genes are tightly associated at the genomic level, an association, however, lost in tetrapods. Our structural and genomic environment comparison reinforces the hypothesis of their phylogenetic relationships with GpH-alpha and -beta. In contrast, the glycosylation status of GPA2 and GPB5 is highly variable further questioning heterodimer secretory efficiency and activity. As a first step toward understanding their function, we investigated the spatiotemporal expression of GPA2 and GPB5 genes at different developmental stages in a basal chordate, the amphioxus. Expression of GPB5 was essentially ubiquitous with an anteroposterior gradient in embryos. GPA2 embryonic and larvae expression was restricted to specific areas and, interestingly, partially overlapped that of a GpH receptor-related gene. In conclusion, we speculate that GPA2 and GPB5 have nondispensable and coordinated functions related to a novelty appeared with bilateria. These proteins would be active during embryonic development in a manner that does not require their heterodimerization.

Pituitary glycoprotein hormone beta subunits in the Australian lungfish and estimation of the relative evolution rate of these subunits within vertebrates.

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.

Molecular characterization of sea bass gonadotropin subunits (alpha, FSHbeta, and LHbeta) and their expression during the reproductive cycle.

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbeta's (35-37%) than to LHbeta's (26-30%), whereas sea bass LHbeta is more similar to LHbeta's (40-53%) than to FSHbeta's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.