The mammalian type II gonadotropin-releasing hormone receptor : cloning, distribution and role in gonadotropin gene expression

Van Biljon, Wilma (2006-12)

Dissertation (PhD)--University of Stellenbosch, 2006.

Thesis

ENGLISH ABSTRACT: Gonadotropin-releasing hormone (GnRH) is well known as the central regulator of the reproductive system through its stimulation of gonadotropin synthesis and release from the pituitary via binding to its specific receptor, known as the gonadotropin-releasing hormone receptor type I (GnRHR-I). The gonadotropins, luteinising hormone (LH) and follicle-stimulating hormone (FSH), bind to receptors in the gonads, leading to effects on steroidogenesis and gametogenesis. The recent finding of a second form of the GnRH receptor, known as the type II GnRHR or GnRHR-II, in non-mammalian vertebrates triggered the interest into the possible existence and function of a GnRHR-II in humans. The current study addressed this issue by investigating the presence of transcripts for a GnRHR-II in various human tissues and cells. While it was demonstrated that antisense transcripts for this receptor, containing sequence of only two of the three coding exons, are ubiquitously and abundantly expressed in all tissues examined, potentially full-length (containing all three exons), sense transcripts for a GnRHR-II were detected only in human ejaculate. Further analysis revealed that the subset of cells in the ejaculate expressing these transcripts is mature sperm. These findings, together with the reported role for GnRH in spermatogenesis and reproduction led to the further analysis of the presence of a local GnRH/GnRHR network in human and vervet monkey ejaculate or sperm. Indeed, such a network seems to be present in humans since transcripts for both forms of GnRH present in mammals, as well as transcripts for the GnRHR-I, are expressed in human ejaculate. Furthermore, transcripts for the GnRHR-II are expressed in both human and vervet monkey ejaculate. Thus, it would appear that locally produced GnRH-1 and/or GnRH-2 in the human male reproductive tract might mediate their effects on fertility via a local GnRHR-I, and possibly via GnRHR-II. Remarkably, in the pituitary, LH and FSH are present in the same gonadotropes, yet they are differentially regulated by GnRH under various physiological conditions. While it is well established that post-transcriptional regulatory mechanisms occur, the contribution of transcriptional regulation to the differential expression of the LHβ- and FSHβ-subunit genes is unclear. In this study, the role of GnRH-1 and GnRH-2 via the GnRHR-I and the GnRHR-II in transcriptional regulation of mammalian LHβ- and FSHβ genes was determined in the LβT2 mouse pituitary gonadotrope cell-line. It is demonstrated for the first time that GnRH-1 may affect gonadotropin subunit gene expression via GnRHR-II in addition to GnRHR-I, and that GnRH-2 also has the ability to regulate gonadotropin subunit gene expression via both receptors. Similar to other reports, it is shown that the transcriptional response to GnRH-1 of LHβ and FSHβ is low (about 1.4-fold for bLHβLuc and 1.2-fold for oFSHβLuc). In addition, evidence is supplied for the first time that GnRH-2 transcriptional regulation of the gonadotropin β subunits is also low (about 1.5-fold for bLHβLuc and 1.1-fold for oFSHβLuc). It is demonstrated that GnRH-1 is a more potent stimulator of bLHβ promoter activity as compared to GnRH-2 via the GnRHR-I, yet both hormones result in a similar maximum induction of bLHβ. However, GnRH-2 is a more efficacious stimulator of bLHβ transcription via the GnRHR-II than GnRH-1. No discriminatory effect of GnRH-1 vs. GnRH-2 was observed for oFSHβ promoter activity via GnRHR-I or GnRHR-II. By comparison of the ratio of expression of transfected oFSHβ- and bLHβ promoterreporters via GnRH-1 with that of GnRH-2, it is shown that GnRH-2 is a selective regulator of FSHβ gene transcription. This discriminatory effect of GnRH-2 is specific for GnRHR-I, as it is not observed for GnRHR-II, where GnRH-1 results in a greater oFSHβ- to-bLHβ ratio. These opposite selectivities for GnRHR-I and GnRHR-II on the ratios of oFSHβ:bLHβ promoter activity for GnRH-1 vs. GnRH-2 suggest a mechanism for fine control of gonadotropin regulation in the pituitary by variation of relative GnRHR-I vs. GnRHR-II levels. In addition, a concentration-dependent modulatory role for PACAP on GnRH-1- and GnRH-2-mediated regulation of bLHβ promoter activity, via both GnRHR-I and GnRHR-II, and of oFSHβ promoter activity, via GnRHR-I, is indicated. The concentration-dependent effects suggest the involvement of two different signalling pathways for the PACAP response. Together these findings suggest that transcription of the gonadotropin genes in vivo is under extensive hormonal control that can be finetuned in response to varying physiological conditions, which include changing levels of GnRH-1, GnRH-2, GnRHR-I and GnRHR-II as well as PACAP.

AFRIKAANSE OPSOMMING: Gonadotropien-vrystellingshormoon (GnRH) is bekend as die sentrale reguleerder van die voorplantingsisteem deur die stimulasie van gonadotropiensintese en - vrystelling vanaf die pituïtêre klier via binding aan ‘n spesifieke reseptor, die sogenaamde tipe I gonadotropien-vrystellingshormoonreseptor (GnRHR-I). Die gonadotropiene, lutineringshormoon (LH) en follikel-stimuleringshormoon (FSH), bind aan reseptore in die gonades waar dit steroïedogenese en gametogenese beïnvloed. Die onlangse ontdekking van ‘n tweede vorm van die GnRH-reseptor, bekend as die tipe II GnRHR of GnRHR-II, in nie-soogdier vertebrate het belangstelling in die moontlike bestaan en funksie van ‘n GnRHR-II in die mens gewek. Hierdie kwessie is aangeraak deur die teenwoordigheid van transkripte vir ‘n GnRHR-II in verskeie weefsel- en seltipes van die mens te ondersoek. Daar is aangetoon dat nie-sin transkripte vir hierdie reseptor, wat die DNA-opeenvolgings van slegs twee van die drie koderende eksons bevat het, oormatig uitgedruk word in al die weefseltipes wat ondersoek is. Daarteenoor is potensieel vollengte (bevattende al drie eksons) sin transkripte vir ‘n GnRHR-II in die mens slegs in semen gevind. Verdere analise het getoon dat dit volwasse sperma binne die semen is wat laasgenoemde transkripte uitdruk. Hierdie bevindinge, tesame met die aangetoonde rol vir GnRH in spermatogenese en reproduksie het gelei tot die verdere analise van die teenwoordigheid van ‘n lokale GnRH/GnRHR-netwerk in mens- en blouaapsemen of -sperm. So ‘n netwerk blyk om teenwoordig te wees in die mens, aangesien transkripte vir beide vorme van GnRH wat in soogdiere gevind word, asook transkripte vir die GnRHR-I, in menssemen uitgedruk word. Daarbenewens word transkripte vir die GnRHR-II uitgedruk in beide mens- en blouaapsemen. Dit wil dus voorkom asof lokaalgeproduseerde GnRH-1 en/of GnRH-2 in die manlike voortplantingstelsel van die mens hul effek op vrugbaarheid bemiddel via ‘n lokale GnRHR-I, en moontlik ook via GnRHR-II. Dit is opmerklik dat LH en FSH teenwoordig is in dieselfde gonadotroopselle van die pituïtêre klier en tog verskillend gereguleer word deur GnRH tydens verskeie fisiologiese kondisies. Terwyl dit bekend is dat post-transkripsionele reguleringsmeganismes teenwoordig is, is die bydrae van transkripsionele regulering tot die differensiële uitdrukking van die LHβ- en FSHβ-subeenheidgene minder duidelik. In hierdie studie is die rol van GnRH-1 en GnRH-2 via die GnRHR-I en die GnRHR-II in transkripsionele regulering van soogdier-LHβ- en -FSHβ-gene in die LβT2 muis pituïtêre gonadotroopsellyn bepaal. Dit is vir die eerste keer aangetoon dat GnRH-1 ‘n effek mag hê op gonadotropiensubeenheid-geenuitdrukking via GnRHR-II bykomend tot GnRHR-I, en dat GnRH-2 ook die vermoë besit om gonadotropiensubeenheid-geenuitdrukking via beide reseptore te reguleer. Soos deur ander studies aangetoon is die transkripsionele respons van LHβ en FSHβ tot GnRH-1 klein (ongeveer 1.4-voudig vir bLHβLuc en 1.2- voudig vir oFSHβLuc). Verder is daar vir die eerste keer bewys gelewer dat transkripsionele regulering van die gonadotropien β-subeenhede deur GnRH-2 ook gering is (ongeveer 1.5-voudig vir bLHβLuc en 1.1-voudig vir oFSHβLuc). Daar is aangetoon dat GnRH-1 ‘n sterker stimuleerder van bLHβ-promotoraktiwiteit is in vergelyking met GnRH-2 via die GnRHR-I, hoewel beide hormone tot ‘n soortgelyke maksimum induksie van bLHβ lei. GnRH-2 is egter ‘n meer effektiewe stimuleerder van bLHβ-transkripsie as GnRH-1 via die GnRHR-II. Geen verskille is gevind tussen die effekte van GnRH-1 en GnRH-2 op oFSHβ-promotoraktiwiteit via GnRHR-I of GnRHR-II nie. Wanneer die verhouding van uitdrukking van getransfekteerde oFSHβ- en bLHβ- promotor-verslaggewers via GnRH-1 met dié van GnRH-2 vergelyk is, is aangetoon dat GnRH-2 ‘n selektiewe reguleerder van FSHβ-geentranskripsie is. Hierdie diskriminasieeffek van GnRH-2 is spesifiek vir GnRHR-I aangesien dit nie vir GnRHR-II waargeneem word nie. GnRH-1 lei tot ‘n groter oFSHβ tot bLHβ-verhouding via GnRHR-II. Hierdie teenoorgestelde selektiwiteite van GnRHR-I en GnRHR-II op die verhoudings van oFSHβ tot bLHβ-promotoraktiwiteit vir GnRH-1 teenoor GnRH-2 suggereer dat daar ‘n meganisme bestaan vir die fyn regulering van gonadotropiene in die pituïtêre klier, deurdat die relatiewe vlakke van GnRHR-I teenoor GnRHR-II gevarieer word. Daarbenewens is ‘n konsentrasie-afhanklike moduleringsrol vir PACAP op GnRH-1- en GnRH-2-bemiddelde regulering van bLHβ-promotoraktiwiteit aangetoon, via beide GnRHR-I en GnRHR-II, asook op oFSHβ-promotoraktiwiteit via GnRHR-I. Hierdie konsentrasie-afhanklike effekte dui op die betrokkenheid van twee verskillende seinpadweë vir die PACAP-respons. Tesame suggereer hierdie bevindinge dat transkripsie van die gonadotropiengene in vivo onder ekstensiewe hormonale kontrole is wat verfyn kan word in respons to veranderlike fisiologiese kondisies. Laasgenoemde sluit veranderende vlakke van GnRH-1, GnRH-2, GnRHR-I en GnRHR-II asook PACAP in.

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