Dissertation (PhD)--University of Stellenbosch, 2006. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17333 |
| Date | 12 1900 |
| Creators | Van Biljon, Wilma |
| Contributors | Hapgood, Janet, University of Stellenbosch. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 638 leaves : ill. (some col.) |
| Rights | University of Stellenbosch |
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